This proposal contains research protocols designed to extend our understanding of the role of oxidative and osmotic stress on lens metabolism. In the long range, it is hoped that this new knowledge will increase our understanding of mechanisms underlying human senile cataract formation and perhaps lead to means of intervening in metabolic sequences to effect a delay or prevention of lens opacification. Human cataracts extracted at the Massachusetts Eye and Ear Infirmary will be collected, photographed and classified by established Cooperative Cataract Research Group (CCRG) techniques. Whole rat and rabbit lenses will also be collected and/or incubated in organ culture and the following studies will be pursued. Adenylate energy change (AEC), phosphorylation potential (PP), [NADPH]/[NADP], [NADH]/[NAD], hexokinase (HK), phosphofructokinase (PFK) and sorbitol pathway activity will be measured in human cataracts, human clear lenses (when available) and animal lenses incubated under conditions creating oxidative stress H202-, -OH and singlet oxygen) and osmotic stress (hyper-and hypolycemia). Regional susceptibility to the above stresses will be determined by studying epithelium, cortex and nuclear fractions. Destructive techniques (i.e. high pressure liquid chromatography and conventional enzyme assays) will be compared to non-destructive techniques (P-31 nuclear magnetic resonance spectroscopy and surface fluorometry) in the analyses of AEC, PP, cofactor ratios and other metobolic intermediates. A new system for photographing human lenses with transmitted light has been developed. Photographs from this system can be analyzed by computerized image analysis thereby yielding a numerical rather than textual cataract classification. This new classification data will supplement the standard CCRG classification data and will be used to establish correlations with the above biochemical data.